U.S. patent application number 12/201467 was filed with the patent office on 2009-03-05 for biosensor chip, process for producing the same, and sensor for surface plasmon resonance analysis.
This patent application is currently assigned to FUJIFILM CORPORATION. Invention is credited to Taisei Nishimi, Yohsuke TAKEUCHI, Hirohiko Tsuzuki.
Application Number | 20090062146 12/201467 |
Document ID | / |
Family ID | 40227533 |
Filed Date | 2009-03-05 |
United States Patent
Application |
20090062146 |
Kind Code |
A1 |
TAKEUCHI; Yohsuke ; et
al. |
March 5, 2009 |
BIOSENSOR CHIP, PROCESS FOR PRODUCING THE SAME, AND SENSOR FOR
SURFACE PLASMON RESONANCE ANALYSIS
Abstract
A biosensor chip includes a substrate, a polymer having an
anionic functional group and being arranged on a surface of the
substrate, a polyamino group which is directly or indirectly bound
to the anionic functional group at a surface of the polymer, and a
long-chain alkyl-based group which is directly or indirectly bound
to the anionic functional group at the surface of the polymer.
Inventors: |
TAKEUCHI; Yohsuke;
(Ashigarakami-gun, JP) ; Nishimi; Taisei;
(Ashigarakami-gun, JP) ; Tsuzuki; Hirohiko;
(Ashigarakami-gun, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W., SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
FUJIFILM CORPORATION
Tokyo
JP
|
Family ID: |
40227533 |
Appl. No.: |
12/201467 |
Filed: |
August 29, 2008 |
Current U.S.
Class: |
506/20 ;
506/30 |
Current CPC
Class: |
B01J 2219/00659
20130101; G01N 2405/00 20130101; B01J 2219/00605 20130101; B01J
2219/00702 20130101; B01J 2219/00527 20130101; B01J 2219/00734
20130101; B01J 2219/00596 20130101; C40B 50/18 20130101; B01J
19/0046 20130101; G01N 33/54373 20130101 |
Class at
Publication: |
506/20 ;
506/30 |
International
Class: |
C40B 40/14 20060101
C40B040/14; C40B 50/14 20060101 C40B050/14 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 29, 2007 |
JP |
2007-222065 |
Claims
1. A biosensor chip comprising: a substrate; a polymer having an
anionic functional group and being arranged on a surface of said
substrate; a first polyamino group which is directly or indirectly
bound to said anionic functional group at a surface of said
polymer; and a long-chain alkyl-based group which is directly or
indirectly bound to said anionic functional group at said surface
of said polymer.
2. A biosensor chip according to claim 1, wherein said first
polyamino group has an acyl group at an end of the first polyamino
group.
3. A biosensor chip according to claim 1, wherein said long-chain
alkyl-based group is bound to said first polyamino group.
4. A biosensor chip according to claim 3, further comprising a
second polyamino group which is directly or indirectly bound to
said polymer.
5. A biosensor chip according to claim 1, wherein said first
polyamino group is a diaminoalkylene group or a di (aminoalkyl)
ether group.
6. A biosensor chip according to claim 2, wherein said first
polyamino group is a diaminoalkylene group or a di (aminoalkyl)
ether group.
7. A biosensor chip according to claim 3, wherein said first
polyamino group is a diaminoalkylene group or a di (aminoalkyl)
ether group.
8. A biosensor chip according to claim 4, wherein said first
polyamino group is a diaminoalkylene group or a di (aminoalkyl)
ether group.
9. A biosensor chip according to claim 1, wherein said long-chain
alkyl-based group is an alkyl-based group having 10 to 22 carbon
atoms.
10. A biosensor chip according to claim 2, wherein said long-chain
alkyl-based group is an alkyl-based group having 10 to 22 carbon
atoms.
11. A biosensor chip according to claim 3, wherein said long-chain
alkyl-based group is an alkyl-based group having 10 to 22 carbon
atoms.
12. A biosensor chip according to claim 4, wherein said long-chain
alkyl-based group is an alkyl-based group having 10 to 22 carbon
atoms.
13. A biosensor chip according to claim 1, wherein said polymer is
carboxymethyl dextran.
14. A biosensor chip according to claim 2, wherein said polymer is
carboxymethyl dextran.
15. A biosensor chip according to claim 3, wherein said polymer is
carboxymethyl dextran.
16. A biosensor chip according to claim 4, wherein said polymer is
carboxymethyl dextran.
17. A biosensor chip according to claim 1, wherein said polymer is
arranged on said substrate through a metal film.
18. A biosensor chip according to claim 17, wherein said metal film
is composed of at least one of metals of gold, silver, copper,
platinum, and aluminum.
19. A biosensor chip according to either of claim 1, wherein said
anionic functional group is a carboxyl group.
20. A process for producing a biosensor chip, comprising the steps
of: (a) activating an anionic functional group in a polymer
arranged on a surface of a substrate; and (b) binding a first
compound containing a polyamino group to said anionic functional
group, and thereafter binding a second compound containing a
long-chain alkyl-based group to said anionic functional group.
21. A process according to either of claim 20, wherein said anionic
functional group is a carboxyl group.
22. A sensor for surface plasmon resonance analysis comprising said
biosensor chip according to claim 1.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a biosensor chip which can
usefully immobilize a bioactive substance, and a process for
producing the biosensor chip. The present invention also relates to
a sensor for surface plasmon resonance analysis.
[0003] 2. Description of the Related Art
[0004] Currently, measurement performed by utilizing interaction
between molecules such as immunoreaction is frequently carried out,
for example, for clinical inspection. Especially, several
techniques enabling highly sensitive detection of variations in the
amount of a material to be detected without bothersome operation or
use of labelled material are currently in practical use. For
example, the SPR (surface plasmon resonance) measurement technique,
the QCM (quartz crystal microbalance) measurement technique, the
technique using the functionalized surfaces of gold particles
having various dimensions in the range from colloidal particles to
ultrafine particles, or other techniques are currently used.
[0005] In the measurement chips for use in measurement in which the
surface plasmon resonance is utilized, an evaporated metal film and
a thin film having a functional group are formed in this order on a
transparent substrate (e.g., a glass substrate), where the
functional group can immobilize a bioactive substance such as a
protein, and the bioactive substance is immobilized on the surface
of the metal film through the functional group. Therefore, it is
possible to analyze interaction between biomolecules by measurement
of a specific binding reaction between the above bioactive
substance and a specimen material.
[0006] The above-mentioned bioactive substance is a biological
macromolecule which is a basic component of a living body (such as
a nucleic acid, a protein, or a polysaccharide), or a constituent
element of a biological macromolecule (such as a nucleotide, a
nucleoside, an amino acid, or a type of sugar), or a material which
controls a living body or changes a function of a living body (such
as a lipid, a vitamin, and a hormone). The bioactive substance is
important, for example, in development of medicine components and
functional foods.
[0007] The lipids are materials which have in a molecule a
long-chain fatty acid or a similar hydrocarbon chain, and have
various functions. For example, the lipids may become an energy
source or a membrane constituent molecule, or may participate in
signal propagation in a cell or nucleus.
[0008] Although the lipids can be classified into the simple
lipids, the complex lipids, and the lipid derivatives, especially,
the phospholipids and the glycolipids are receiving attention in
connection with carbohydrate metabolism. Although the phospholipids
are insoluble in water, the phospholipids form micelles which are
insoluble in water since the phospholipids have a molecular
structure containing a polar group and a nonpolar group and being
amphiphilic. The phosphoric acid in a phospholipid is hydrophilic.
Therefore, when a phospholipid comes in contact with a solvent, the
phospholipid can form a liposome (a lipid membrane vesicle), which
is a soluble micelle formed with an artificial phospholipid
membrane.
[0009] Since the phospholipids have the above property, the
phospholipids are currently being used in study of a biomembrane
model, and the use of the phospholipids as materials in drug
delivery systems are currently proceeding toward commercialization
and are being actively studied.
[0010] For example, the Biacore L1 chip is known as a chip which
can trap lipids, liposomes, and the like. In the Biacore L1 chip,
dextran with which a substrate is coated is modified with a
long-chain alkane, so that lipid liposomes and the like can be
absorbed by the long-chain alkane. (Biacore is a trademark of GE
Healthcare Companies.) See M. A. Cooper et al., "A Vesicle Capture
Sensor Chip for Kinetic Analysis of Interactions with
Membrane-Bound Receptors," Analytical Biochemistry, Vol. 277, Issue
2, pp. 196-205, 2000, and E. M. Erb et al., "Characterization of
the Surfaces Generated by Liposome Binding to the Modified Dextran
Matrix of a Surface Plasmon Resonance Sensor Chip," Analytical
Biochemistry, Vol. 280, Issue 1, pp. 29-35, 2000.
[0011] However, since the Biacore L1 chip has the carboxyl group,
which is anionic, the Biacore L1 chip cannot absorb anionic or
nonionic lipids or liposomes although the Biacore L1 chip can
efficiently absorb cationic lipids or liposomes.
SUMMARY OF THE INVENTION
[0012] The present invention has been made in view of the above
circumstances.
[0013] The first object of the present invention is to provide a
biosensor chip which can immobilize a bioactive substance on a
surface of the biosensor chip without influence of electric charge
repulsion.
[0014] The second object of the present invention is to provide a
process for producing a biosensor chip which achieves the first
object.
[0015] The third object of the present invention is to provide a
sensor for surface plasmon resonance analysis, where the sensor
uses the biosensor chip which achieves the first object.
[0016] (I) In order to accomplish the first object, according to
the first aspect of the present invention, a biosensor chip is
provided. The biosensor chip according to the first aspect of the
present invention comprises a substrate; a polymer having an
anionic functional group and being arranged on a surface of the
substrate; a first polyamino group which is directly or indirectly
bound to the anionic functional group at a surface of the polymer;
and a long-chain alkyl-based group which is directly or indirectly
bound to the anionic functional group at the surface of the
polymer.
[0017] The expression "directly bound to the to the anionic
functional group" means to be directly bound to the anionic
functional group without a linking group, and the expression
"indirectly bound to the anionic functional group" means to be
indirectly bound to the anionic functional group through a linking
group. The linking group may be derived from the polymer, or may be
bound to the polymer for binding the first polyamino group or the
long-chain alkyl-based group to the polymer.
[0018] The long-chain alkyl-based group is preferably an alkyl
chain containing 10 to 22 carbon atoms, and more preferably an
alkyl chain containing 12 to 18 carbon atoms. In some cases, one or
more heteroatoms may exist between sequences of carbon atoms
constituting the long-chain alkyl-based group, and/or one or more
double bonds and/or one or more triple bonds may exist between
single-bonded sequences of carbon atoms constituting the long-chain
alkyl-based group. In addition, it is preferable that the
long-chain alkyl-based group have a normal-chain structure (which
is not branched).
[0019] The anionic functional group is typically the carboxyl
group.
[0020] In addition, preferably, the biosensor chip according to the
first aspect of the present invention may further have one or any
possible combination of the following additional features (i) to
(vii).
[0021] (i) It is preferable that the first polyamino group have an
acyl group at an end of the first polyamino group.
[0022] (ii) The long-chain alkyl-based group may be bound to the
first polyamino group. In this case, the biosensor chip may further
comprise a second polyamino group which is directly or indirectly
bound to the polymer.
[0023] (iii) It is preferable that the first polyamino group be a
diaminoalkylene group or a di(aminoalkyl) ether group.
[0024] (iv) It is preferable that the long-chain alkyl-based group
is an alkyl-based group having 10 to 22 carbon atoms.
[0025] (v) It is preferable that the polymer is carboxymethyl
dextran.
[0026] (vi) It is preferable that the polymer is arranged on the
substrate through a metal film.
[0027] (vii) It is preferable that the metal film is composed of at
least one of the metals of gold, silver, copper, platinum, and
aluminum.
[0028] (II) In order to accomplish the second object, according to
the second aspect of the present invention, a process for producing
a biosensor chip is provided. The process according to the second
aspect of the present invention comprises the steps of: (a)
activating an anionic functional group in a polymer arranged on a
surface of a substrate; and (b) binding a first compound containing
a polyamino group to the anionic functional group, and thereafter
binding a second compound containing a long-chain alkyl-based group
to the anionic functional group.
[0029] (III) In order to accomplish the third object, according to
the third aspect of the present invention, a sensor for surface
plasmon resonance analysis comprising the biosensor chip according
to the first aspect of the present invention is provided. That is,
the biosensor chip according to the first aspect of the present
invention can be preferably used as a sensor chip in a sensor for
surface plasmon resonance analysis.
[0030] (IV) The advantages of the present invention are explained
below.
[0031] Since the biosensor chip according to the first aspect of
the present invention comprises the polymer having an anionic
functional group (e.g., the carboxyl group) and being arranged on a
surface of the substrate, and a polyamino group directly or
indirectly bound to the anionic functional group at a surface of
the polymer, and a long-chain alkyl-based group directly or
indirectly bound to the anionic functional group at the surface of
the polymer, the long-chain alkyl-based group can stably immobilize
a bioactive substance on the surface of polymer, and the polyamino
group can cancel the electric charge of the anionic functional
group in the polymer. Therefore, the biosensor chip according to
the first aspect of the present invention can immobilize the
bioactive substance on the surface of the polymer without influence
of the electric charge repulsion. In addition, although the
nonspecific absorption can be frequently caused by electric charge,
the biosensor chip according to the first aspect of the present
invention can reduce the nonspecific absorption while the bioactive
substance is immobilized.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 is a diagram schematically illustrating an
arrangement of chemical components in a first embodiment of the
biosensor chip according to the present invention.
[0033] FIG. 2 is a diagram schematically illustrating an
arrangement of chemical components in a second embodiment of the
biosensor chip according to the present invention.
[0034] FIG. 3 is a diagram schematically illustrating an
arrangement of chemical components in a third embodiment of the
biosensor chip according to the present invention.
[0035] FIG. 4 is a diagram schematically illustrating a
configuration of a sensor for surface plasmon resonance analysis,
which comprises the biosensor chip according to the present
invention.
[0036] FIG. 5 is a sensorgram of the biosensor chip according as a
concrete example 1 of the biosensor chip according to the present
invention.
[0037] FIG. 6 is a sensorgram of a conventional biosensor chip as a
comparison example.
DETAILED DESCRIPTION OF THE INVENTION
[0038] Preferred embodiments of the present invention are explained
in detail below with reference to drawings.
1. Biosensor Chips
1.1 Structures of Producing Biosensor Chips
[0039] Hereinbelow, the biosensor chips according to the first to
third embodiments of the present invention are explained below with
reference to FIGS. 1 to 3, which schematically show the
arrangements of chemical components in the first to third
embodiments.
[0040] As illustrated in FIG. 1, the biosensor chip according to
the first embodiment comprises a substrate, a polymer having the
carboxyl group (as the aforementioned anionic functional group) and
being arranged on a surface of the substrate, a long-chain
alkyl-based group which is bound to the surface of the polymer
through a group R.sup.1, and a polyamino group which has an acyl
group at en end of the polyamino group and is bound to the surface
of the polymer through a group R.sup.2. In the biosensor chip
according to the first embodiment, the polyamino group can cancel
the electric charge of the carboxyl group in the polymer, and the
long-chain alkyl-based group can stably immobilize a bioactive
substance on the surface of polymer.
[0041] As illustrated in FIG. 2, the biosensor chip according to
the second embodiment comprises a substrate, a polymer having the
carboxyl group (as the aforementioned anionic functional group) and
being arranged on a surface of the substrate, a polyamino group
which is bound to the surface of the polymer through the group
R.sup.2, and a long-chain alkyl-based group which is bound to the
polyamino group through a group R.sup.3. In the biosensor chip
according to the second embodiment, the polyamino group can cancel
the electric charge of the carboxyl group in the polymer, and the
long-chain alkyl-based group can stably immobilize a bioactive
substance on the surface of polymer.
[0042] As illustrated in FIG. 3, the biosensor chip according to
the third embodiment comprises a substrate, a polymer having the
carboxyl group (as the aforementioned anionic functional group) and
being arranged on a surface of the substrate, a first polyamino
group which is bound to the surface of the polymer through the
group R.sup.2, a long-chain alkyl-based group which is bound to the
first polyamino group through the group R.sup.3, and a second
polyamino group which has an acyl group at en end of the second
polyamino group and is bound to the surface of the polymer through
the group R.sup.2. In the biosensor chip according to the third
embodiment, the polyamino group can cancel the electric charge of
the carboxyl group in the polymer, and the long-chain alkyl-based
group (bound through the linking groups) can stably immobilize a
bioactive substance on the surface of polymer.
[0043] As mentioned before, the biosensor chips according to the
first to third embodiments basically comprise a polymer having the
carboxyl group (as the aforementioned anionic functional group) and
being arranged on a surface of a substrate, a polyamino group which
is directly or indirectly bound to the surface of the polymer, and
a long-chain alkyl-based group which is directly or indirectly
bound to the surface of the polymer. Although, in the biosensor
chips illustrated in FIGS. 1 to 3, the long-chain alkyl-based group
is indirectly bound to the surface of the polymer through the group
R.sup.1, and the polyamino group is also indirectly bound to the
surface of the polymer through the group R.sup.2, the long-chain
alkyl-based group or the polyamino group can be directly bound to
the carboxyl group in the polymer. Therefore, the groups R.sup.1
and R.sup.2 can be dispensed with. In addition, although, in the
biosensor chip illustrated in FIG. 2, the long-chain alkyl-based
group is indirectly bound to the polyamino group through the group
R.sup.3, the group R.sup.3 can be dispensed with in the case where
one or both of the long-chain alkyl-based group and the polyamino
group have a functional group behaving as a linking group which can
bind the long-chain alkyl-based group and the polyamino group.
[0044] It is necessary to couple an acyl group to an open end of
the polyamino group in order to suppress nonspecific absorption
which can occur at the open end of the polyamino group. However, in
the case where the long-chain alkyl-based group is bound to the
polyamino group (as in the biosensor chips of FIGS. 2 and 3), the
acyl group can be dispensed with since the long-chain alkyl-based
group suppresses the nonspecific absorption.
[0045] In the biosensor chips of FIGS. 1, 2, and 3, the group
R.sup.1 is preferably the aminocarbonyl group, the carbamoyl group,
the oxycarbonyl group, the carbonyloxy group, the carbonyl group,
the ether group, the thioether group, or the like, the group
R.sup.2 is preferably the carbamoyl group, the carbonyloxy group,
the carbonyl group, or the like, and the group R.sup.3is preferably
the carbamoyl group, the carbonyloxy group, the carbonyl group, or
the like. Especially, from the viewpoint of the reactivity and the
binding stability, it is further preferable that the group R.sup.1
be the aminocarbonyl group or the carbamoyl group, the group
R.sup.2 be the carbonyl group, and the group R.sup.3 be the
carbonyl group.
[0046] The long-chain alkyl-based group is preferably an alkyl
chain containing 10 to 22 carbon atoms, and more preferably an
alkyl chain containing 12 to 18 carbon atoms. In some cases, one or
more heteroatoms may exist between sequences of carbon atoms
constituting the long-chain alkyl-based group, and/or one or more
double bonds and/or one or more triple bonds may exist between
single-bonded sequences of carbon atoms constituting the long-chain
alkyl-based group. In addition, it is preferable that the
long-chain alkyl-based group have a normal-chain structure (which
is not branched). Specifically, preferable examples of the
long-chain alkyl-based group are the lauryl group, the myristyl
group, the cetyl group, the stearyl group, the arachidyl group, the
behenyl group, the oleyl group, and the like. Among all, the
stearyl group and the oleyl group are particularly preferable since
the stearyl group and the oleyl group can realize both of simple
modification reaction and absorption of lipids.
[0047] Each atomic group realizing the polyamino group contains
preferably two to four amino groups, and more preferably two or
three amino groups. Specifically, the diaminoalkylene groups and
the di (aminoalkyl) ether groups can be used as the polyamino
group. More specifically, preferable examples of the polyamino
group are the following aliphatic diamine groups, aromatic diamine
groups, and polyamine groups. The aliphatic diamine groups include
the ethylenediamine group, the tetraethylenediamine group, the
octamethylenediamine group, the decamethylenediamine group, the
piperazine group, the triethylenediamine group, the
diethylenetriamine group, the triethylenetetramine group, the
dihexamethylenetriamine group, the 1,4-diaminocyclohexane group,
and the like. The aromatic diamine groups include the
paraphenylenediamine group, the methaphenylenediamine group, the
paraxylylenediamine group, the methaxylylenediamine group, the
4,4'-diaminobiphenyl group, the 4,4'-diaminodiphenylmethane group,
the 4,4'-diaminodiphenylketone group, the
4,4'-diaminodiphenylsulfonic acid group, and the like. The
polyamine groups include the diethylenetriamine group, the
triethylenetetramine group, the tetraethylenepentamine group, the
pentaethylenehexamine group, the spermidine group, the spermine
group, the polyethyleneimine, and the like. Especially, from the
viewpoint of water solubility and high reactivity in the
modification reaction, the ethylenediamine group, the
di(2-aminoethyl) ether group, and the di (3-aminopropyl) ether
group can be preferably used.
[0048] The acyl group is generally expressed by the formula
R.sup.4CO--, and the group R.sup.4maybe discontinued by
substitution with a heteroatom in some cases. The group R4 is
preferably an alkyl chain containing 1 to 21 carbon atoms, and more
preferably an alkyl chain containing 1 to 11 carbon atoms. In
addition, the group R.sup.4 preferably has a normal-chain structure
(which is not branched), and may be a hydrocarbon chain containing
one or more doubles bond and/or one or more triple bonds in some
cases. Specifically, preferable examples of the acyl group are the
formyl group, the acetyl group, the propanoyl group, the
isopropanoyl group, the butanoyl group, the lauryl group, the
myristyl group, the stearyl group, the oleyl group, and the like.
Especially, from the viewpoint of reactivity, the formyl group, the
acetyl group, and the propanoyl group can be preferably used.
1.2 Process for Producing Biosensor Chip
[0049] The biosensor chips according to the first to third
embodiments can be produced by activating the carboxyl group
(contained in the polymer arranged on the substrate) in a similar
manner to the activation of a polymer bound to a substrate which is
coated with a self-assembled film (as explained later), binding a
compound having a polyamino group (i.e., a polyamino compound) to
the activated carboxyl group, and thereafter binding a compound
having a long-chain alkyl-based group. Since the compound having
the long-chain alkyl-based group is bound after the compound having
the polyamino group is bound, it is possible to easily introduce
the long-chain alkyl-based group, and reduce the positive electric
charge on the surface of the polymer by improving the reaction
efficiency.
[0050] For example, the biosensor chip according to the first
embodiment can be produced by activating carboxyl groups on the
polymer, causing a reaction of a polyamino compound with first part
of the carboxyl groups, reactivating second part of the carboxyl
groups which are not activated, causing a reaction of a compound
having a long-chain alkyl-based group with the second part of the
carboxyl groups, and finally performing acetylation. The
acetylation is a process for attaching an acyl group onto the chip.
The acyl group can be attached onto the chip by causing a reaction
with the acyl group which is activated as a derivative such as acyl
chloride or an acid anhydride. Alternatively, the biosensor chip
according to the first embodiment can also be produced by
reactivating the second part of the carboxyl groups which are not
activated, and causing a direct reaction of a polyamino compound
having an acyl group at an end of the polyamino compound, with the
second part of the carboxyl groups reactivated as above.
[0051] The biosensor chip according to the second embodiment can be
produced by activating the carboxyl group at the surface of the
polymer, binding a reaction of a polyamino compound with the
carboxyl group, and causing a reaction of a compound having a
long-chain alkyl-based group with the polyamino compound.
[0052] The biosensor chip according to the third embodiment can be
produced by activating the carboxyl group on the polymer, causing a
reaction with a first polyamino compound and a reaction with a
second polyamino compound having an acyl group at an end of the
second polyamino compound, activating the first polyamino compound,
and causing a reaction of a compound having a long-chain
alkyl-based group with the first polyamino compound.
[0053] In the above processes for producing the biosensor chips
according to the first to third embodiments, the polyamino compound
may be a diaminoalkylene or a di (aminoalkyl) ether. Specifically,
the polyamino compound is one of the following aliphatic diamines,
aromatic diamines, and polyamines. The aliphatic diamines include
ethylenediamine, tetraethylenediamine, octamethylenediamine,
decamethylenediamine, piperazine, triethylenediamine,
diethylenetriamine, triethylenetetramine, dihexamethylenetriamine,
1,4-diaminocyclohexane, and the like. The aromatic diamines include
paraphenylenediamine, methaphenylenediamine, paraxylylenediamine,
methaxylylenediamine, 4,4'-diaminobiphenyl,
4,4'-diaminodiphenylmethane, 4,4'-diaminodiphenylketone,
4,4'-diaminodiphenylsulfone, and the like. The polyamines include
diethylenetriamine, triethylenetetramine, tetraethylenepentamine,
pentaethylenehexamine, spermidine, spermine, polyethyleneimine, and
the like. Especially, from the viewpoint of water solubility and
high reactivity in modification reaction, ethylenediamine,
di(2-aminoethyl) ether, and di(3-aminopropyl) ether can be
preferably used.
[0054] The compound having a long-chain alkyl-based group is
preferably a fatty acid which contains an alkyl chain having 10 to
22 carbon atoms, and is more preferably a fatty acid which contains
an alkyl chain having 12 to 18 carbon atoms. In some cases, one or
more heteroatoms may exist between sequences of carbon atoms
constituting the long-chain alkyl-based group, and/or one or more
double bonds and/or one or more triple bonds may exist between
single-bonded sequences of carbon atoms constituting the long-chain
alkyl-based group. In addition, it is preferable that the
long-chain alkyl-based group have a normal-chain structure (which
is not branched). Specifically, preferable examples of the fatty
acid are lauric acid, myristic acid, palmitic acid, stearic acid,
arachidic acid, behenic acid, oleic acid, and the like. Among all,
stearic acid and oleic acid are particularly preferable since
stearic acid and oleic acid can realize both of simple modification
reaction and absorption of lipids.
[0055] The polyamino compound having an acyl group at an end is a
monoacylalkyldiamine, a monoacylalkylenediamine, a
diacylalkyltriamine, or the like. Preferable examples of the
polyamino compound having an acyl group at an end are
monostearoylethylenediamine, monooleoylethylenediamine,
monopalmitoylethylenediamine, monolauroylethylenediamine,
monostearoylphenylenediamine, monooleoylphenylenediamine,
monopalmitoylphenylenediamine, monolauroylphenylenediamine,
1,2,-oleoylpropanetriamine, monostearoyl (diaminoethyl ether), and
monooleoyl(diaminoethyl ether). Among all,
monostearoylethylenediamine, monooleoylethylenediamine,
monostearoyl(diaminoethyl ether), and monooleoyl(diaminoethyl
ether) are particularly preferable.
[0056] The above compounds can be attached onto the chip by causing
a reaction with a reactive group which is activated by a process
for activating a polymer as explained later.
1.3 Constituents of Biosensor Chip
[0057] Next, details of the substrate, the polymer, and the like,
which constitute the biosensor chips according to present
invention, are explained below.
1.3.1 Substrate and Metal Film
[0058] For example, in the case where the biosensor chip according
to the present invention is to be used in a surface plasmon
resonance biosensor, a substrate of a material transparent to laser
light, such as optical glass (e.g., the type BK7 glass) or
synthetic resin (e.g., polymethyl methacrylate, polyethylene
terephthalate, polycarbonate, or a cycloolefin polymer) can be used
as the substrate in the biosensor chip according to the present
invention. It is preferable that the material of the substrate not
be anisotropic to polarized light, and be superior in machinability
or processibility.
[0059] In the above case, a metal film is arranged over the
substrate. At this time, the metal film may be arranged in direct
contact with the substrate, or over the substrate through another
layer. The composition of the metal film is not specifically
limited as long as the surface plasmon resonance can occur.
However, it is preferable that the substrate be made of one or a
combination of gold, silver, copper, platinum, palladium, and
aluminum, and is particularly preferable that the substrate be made
of gold. In addition, it is possible to arrange an interposing
layer of chromium or the like between the substrate and the metal
film.
[0060] Although the thickness of the metal film is not specifically
limited, the thickness of the metal film is preferably 0.1 to 500
nm, and particularly preferably 1 to 200 nm. In the case where the
thickness of the metal film exceeds 500 nm, it is impossible to
sufficiently detect the surface plasmon resonance of a medium. In
the case where the interposing layer of chromium or the like is
arranged, the thickness of the interposing layer is preferably 0.1
to 10 nm.
[0061] The metal film may be formed by one of the conventional
film-formation techniques such as sputtering, evaporation, ion
plating, electroplating, and nonelectrolytic plating.
1.3.2 Polymer
[0062] The polymer (polymer film) is bound to the substrate through
the metal film. The polymer film can be constituted by one or
combination of hydrophilic polymers and hydrophobic polymers.
However, it is preferable to bind a hydrophilic polymer from the
viewpoint that bioactive substances can be three-dimensionally
bound to the hydrophilic polymer. Although the polymer film may be
bound to the substrate either directly or indirectly, it is
preferable that the polymer film be formed on a self-assembled
film.
[0063] The polymer in each of the biosensor chips according to the
first to third embodiments has the carboxyl group. The carboxyl
group may be originally contained in the polymer, or may be added
to a polymer which does not originally contain the carboxyl group.
The carboxyl group can be added by ozonization, plasma processing,
hydrolysis of the polymer by alkali such as NaOH, binding of a
linker having the carboxyl group, or another technique.
[0064] In addition, even in the biosensor chips in which the
polymer contains another functional group having the negative
electric charge such as the sulfonic acid (sulfo) group or the
sulfinic group, instead of the carboxyl group, the negative
electric charge at the surface of the polymer can also be cancelled
by binding a polyamino group to the polymer. Therefore, such
biosensor chips can also achieve the advantages similar to the
present invention.
1.3.3 Self-Assembled Film
[0065] The self-assembled film is an ultrathin film (such as a
monomolecular film or an LB (Langmuir-Blodgett) film) which is
formed into an ordered structure by a self-assembling mechanism
possessed by the material of the film per se. The self-assembling
mechanism enables formation of an ordered structure or pattern over
a wide area under a non-equilibrium condition.
[0066] It is preferable that the self-assembled film be formed of a
compound expressed by a constitutional formula X--R.sup.5--Y, where
the X represents a group which can be bound to the metal film,
R.sup.5 represents a divalent organic linking group, and Y
represents a group which can be bound to hydrophilic polymers, NTA
(nitrilotriacetic acid), and the like. Specifically, the group X is
--SH, --SS, --SeH, --SeSe, --COSH, or the like, and the group Y is
one of the functional groups of the hydroxy group, the
hydroxycarbonyl group, alkoxy groups, and alkyl groups. In some
cases, one or more heteroatoms may exist between sequences of
carbon atoms constituting the divalent organic linking group
R.sup.5. Preferably, the divalent organic linking group R.sup.5 is
a normal (unbranched) organic chain structure suitable for
desirably dense packing. In some cases, the divalent organic
linking group R.sup.5 is a hydrocarbon chain having one or more
double bonds and/or one or more triple bonds, or the hydrocarbon
chain may be perfluorinated. It is preferable that the linking
group R5 has a length corresponding to an alkyl chain containing 2
to 8 carbon atoms.
[0067] Further specifically, the self-assembled film can be
preferably formed, on the metal film, of alkanethiols in the case
where the metal film is formed of gold, alkylsilanes in the case
where the metal film is formed of glass, and alcohols in the case
where the metal film is formed of silicon. Specific examples of the
alkanethiols which can be used for the self-assembled film are
7-carboxy-1-heptanethiol, 10-carboxyl-1-decanethiol,
4,4'-dithiodibutyric acid, and 11-hydroxy-1-undecanethiol, and
11-amino-1-undecanethiol. Specific examples of the alkylsilanes are
aminopropyltrimethoxysilane, aminoethylaminotriethoxysilane,
hydroxypropyltriethoxysilane, and the like.
1.3.4 Hydrophilic Polymer
[0068] The natural polymers such as dextran derivatives, starch
derivatives, cellulose derivatives, and gelatines and the synthetic
polymers such as polyvinyl alcohol, polyethylene glycol,
polyvinylpyrrolidone, polyacrylamide derivatives, and
polymethylvinyl ether are examples of the hydrophilic polymer.
[0069] In addition, synthetic polymers containing the carboxyl
group and natural polymers containing the carboxyl group can be
used as the polymer containing the carboxyl group. The synthetic
polymers containing the carboxyl group include polyacrylic acid,
polymethacrylic acid, and copolymers of polyacrylic acid and
polymethacrylic acid. For example, the synthetic polymers
containing the carboxyl group include the methacrylic acid
coplymer, the acrylic acid coplymer, the itaconic acid coplymer,
the crotonic acid coplymer, the maleic acid coplymer, the partially
esterificated maleic acid coplymer, and the polymer produced by
adding an acid anhydride to a polymer containing the hydroxy group,
which are disclosed in Japanese Examined Patent Publication No.
59(1984)-044615 (corresponding to U.S. Pat. No. 4,139,391),
Japanese Examined Patent Publication No. 54(1979) -034327
(corresponding to U.S. Pat. No. 3,804,631), Japanese Examined
Patent Publication No. 58(1983)-012577 (corresponding to U.S. Pat.
No. 3,930,865), Japanese Examined Patent Publication No.
54(1979)-025957 (corresponding to British Patent Publication No. 1
521 372), Japanese Unexamined Patent Publication No.
59(1984)-053836 (corresponding to U.S. Pat. No. 4,687,727), and
Japanese Unexamined Patent Publication No. 59(1984)-071048
(corresponding to U.S. Pat. No. 4,537,855).
[0070] The natural polymers containing the carboxyl group may be
extracts from natural plants or products of microorganism
fermentation, enzyme synthesis, and chemical synthesis. The natural
polymers containing the carboxyl group include polysaccharides such
as hyaluronic acid, chondroitin sulfate, heparin, dermatan sulfate,
carboxymethylcellulose, carboxyethylcellulose, cellouronic acid,
carboxymethyl chitin, carboxymethyl dexetran, and carboxymethyl
starch, and polyamino acids such as polyglutaminic acid and
polyaspartic acid. The polysaccharides containing the carboxyl
group are commercially available. For example, carboxymethyl
dexetran is available as the products CMD, CMD-L, and CMD-D40 from
Meito Sangyo Co., Ltd, carboxymethylcellulose is available in the
form of sodium carboxymethylcellulose from Wako Pure Chemical
Industries, Limited, and alginic acid is available in the form of
sodium alginate from Wako Pure Chemical Industries, Limited.
[0071] The polymer containing the carboxyl group used in the
biosensor chip according to the present invention is preferably one
of polysaccharides containing the carboxyl group, and more
preferably carboxymethyl dexetran.
[0072] The molecular weight of the polymer containing the carboxyl
group used in the biosensor chip according to the present invention
is not specifically limited. However, the average molecular weight
of the polymer containing the carboxyl group is preferably 1,000 to
5,000,000, more preferably 10,000 to 2,000,000, and further
preferably 100,000 to 1,000,000. When the average molecular weight
of the polymer containing the carboxyl group is smaller than the
range of 1,000 to 5,000,000, the immobilized amount of the
bioactive substance becomes too small. On the other hand, when the
average molecular weight of the polymer containing the carboxyl
group is greater than the range of 1,000 to 5,000,000, high
viscosity of the polymer solution makes the handling of the polymer
solution difficult.
[0073] The hydrophilic polymer as described above may be bound to
the substrate through the aforementioned self-assembled film or
hydrophobic polymer, or may be formed directly on the substrate by
use of a solution containing the polymer. In addition, the
hydrophilic polymer may be bridged.
[0074] In the case where the hydrophobic polymer is used,
preferable examples of the hydrophobic polymer are polyacrylic acid
derivatives, polymethacrylic acid derivatives, polyethylene (PE),
polypropylene (PP), polybutadiene, polymethylpentene, cycloolefin
polymer, polystyrene (PS), acrylonitrile/butadiene/styrene
copolymer (ABS), styrene/maleic anhydride copolymer/polyvinyl
chloride (PVC), polyethylene terephthalate (PET), polyethylene
naphthalate (PEN), nylon 6, nylon 66, cellulose acetate (TAC),
polycarbonate (PC), modified polyphenylene ether (m-PPE),
polyphenylene sulfide (PPS), polyether ketone (PEK), polyether
ether ketone (PEEK), polysulfone (PSF), polyether sulfone (PES),
polyphenylene sulfide (PPS), and liquid crystal polymers (LCP).
Further, the above hydrophobic polymers can turn into a hydrophilic
polymer when cationic groups are introduced into the hydrophobic
polymer at a high rate.
[0075] The substrate can be coated with hydrophobic polymer by the
conventional techniques such as spin coating, air-knife coating,
bar coating, blade coating, slide coating, curtain coating,
spraying, evaporation, casting, dipping (immersion), or the
like.
1.3.5 Activation of Polymer
[0076] In the case where the hydrophilic polymer is used as the
polymer containing the carboxyl group, and the substrate is coated
with a self-assembled film, the polymer can be bound to the
substrate by activating the carboxyl group. The polymer containing
the carboxyl group can be preferably activated, for example, by
using the following conventional techniques (1) to (3).
[0077] (1) The activation by use of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and
N-hydroxysuccinimide (NHC), which are water-soluble
carbodiimides
[0078] (2) The technique which is disclosed in Japanese Unexamined
Patent Publication No. 2006-058071, and in which the carboxyl group
is activated by use of one of the uronium salt, the phosphonium
salt, and the triazine derivatives which has a specific
structure
[0079] (3) The technique which is disclosed in Japanese Unexamined
Patent Publication No. 2006-090781, and in which the carboxyl group
is activated by processing using a triazine derivative or a salt of
the triazine derivative, and subsequent processing using one of a
nitrogen-containing hetero aromatic compound having the hydroxy
group, a phenol derivative having an electron attracting group, and
an aromatic compound having the thiol group
[0080] Although the polymer containing the activated carboxyl group
can be bound to the substrate by causing a reaction of a solution
of the polymer with the substrate, it is preferable to cause the
reaction after a thin film is formed on the substrate by spin
coating or the like.
[0081] After the carboxyl group in the polymer is activated as
above, a reaction of a compound having the polyamino group with the
polymer and a reaction of a compound having the long-chain
alkyl-based group are caused. Thus, production of the biosensor
chips according to the embodiments can be completed.
1.4 Bioactive Substance
[0082] The bioactive substance maybe, for example, an
immunoprotein, an enzyme, a microorganism, and a nucleic acid, a
low-molecular organic compound, a nonimmune protein, an
immunoglobulin-binding protein, a sugar-binding protein, a sugar
chain which recognizes a type of sugar, a lipid, a fatty acid, a
fatty acid ester, or a polypeptide or an oligopeptide having
ligand-binding ability. The bioactive substance can be immobilized
by application of or immersion in a solution containing the
bioactive substance.
2. Biosensor
[0083] The scope of the biosensor of the present invention should
be considered in the broadest meaning, and the biosensor of the
present invention is a sensor which detects and measures an
objective substance by converting an interaction between
biomolecules into a signal such as an electric signal. Normally,
the biosensor chip is constituted by a receptor part and a
transducer part. The receptor part recognizes a chemical substance
to be detected, and the transducer part coverts a physical or
chemical change occurring in the receptor part into an electric
signal. Living bodies contain such pairs of substances that the
substances (in each pair) have an affinity for each other. Examples
of such pairs are an enzyme and a substrate, an enzyme and a
coenzyme, an antigen and an antibody, or a hormone and a receptor.
When one of substances having an affinity for the other is fixed as
a molecular recognizer to a substrate, the biosensor can
selectively measure the other of the substances.
[0084] The biosensor chip according to the present invention can be
used for detection and/or measurement of an interaction between a
substance to be examined and a bioactive substance immobilized to
the substrate of the biosensor chip. As mentioned before, the
biosensor chip according to the present invention can be used in a
biosensor performing one of the surface plasmon resonance (SPR)
measurement, the quartz crystal microbalance (QCM) measurement, and
the measurement using the functionalized surfaces of gold particles
having various dimensions in the range from colloidal particles to
ultrafine particles, and the like.
[0085] Preferably, the biosensor chip is used as a biosensor chip
for surface plasmon resonance analysis, and a metal film is
arranged on a transparent substrate. Although, generally, the
biosensor chip for surface plasmon resonance analysis comprises a
member containing a first part in which irradiation light
propagates and reflects and a second part on which a bioactive
substance is to be immobilized, the biosensor chip according to the
present invention can be used as a member having the second part on
which a bioactive substance is to be immobilized.
[0086] An example of a surface-plasmon-resonance measurement system
which analyzes a property of a substance to be measured uses a
system called the Kretschmann configuration. (See, for example,
Japanese Unexamined Patent Publication No. 6(1994)-167443.)
Basically, the above surface-plasmon-resonance measurement system
is constituted by a dielectric block, a metal film, a light source,
an optical system, and an optical detection means. The dielectric
block is formed, for example, in the form of a prism. The metal
film is formed on a face of the dielectric block so as to be in
contact with a substance to be measured (such as a specimen
solution). The light source generates a light beam. The optical
system can make the light beam inject onto the dielectric block at
various incident angles so as to satisfy the total reflection at
the interface between the dielectric block and the metal film. The
optical detection means detects the surface-plasmon-resonance
state, i.e., the attenuated total reflection (ATR)), by measuring
the intensity of a portion of the light beam which is reflected at
the above interface.
[0087] Hereinbelow, an embodiment of a sensor for surface plasmon
resonance analysis according to the present invention is explained
with reference to FIG. 4, which is a schematic side view
illustrating a configuration of the sensor for surface plasmon
resonance analysis, where the sensor comprises the biosensor chip
according to the present invention. The sensor comprises a
plurality of measurement units and a display unit 21. Each
measurement unit comprises a biosensor chip 10, a laser-light
source 14, an incident-beam optical system 15, a collimator lens
16, an optical detector 17, a differential-amplifier array 18, a
driver 19, and a signal processing unit 20. The laser-light source
14 generates a light beam 13. The incident-beam optical system 15
makes the light beam 13 injected into the biosensor chip 10. The
collimator lens 16 collimates the light beam 13 reflected by the
biosensor chip 10, and outputs the reflected light beam 13 toward
the optical detector 17. The optical detector 17 receives the
reflected light beam 13 from the biosensor chip 10, and detects the
intensity of the reflected light beam 13. The
differential-amplifier array 18 is connected to the optical
detector 17, and the driver 19 is connected to the
differential-amplifier array 18. The signal processing unit 20 is
realized by a computer system or the like, and connected to the
driver 19. The signal processing unit 20 has a function of
processing the signal outputted from the optical detector 17
through the differential-amplifier array 18 and the driver 19, and
a function of correcting the sensitivity of the biosensor chip 10.
That is, the signal processing unit 20 behaves as a sensitivity
correction means as well as a signal processing means. The optical
detector 17, the differential-amplifier array 18, the driver 19,
and the signal processing unit 20 realize a measurement means for
measuring the position of a dark line in the reflected light beam
13.
[0088] The biosensor chip 10 is constituted by a dielectric block
11 and a metal film 12. The dielectric block 11 has a shape
produced by removing from a pyramid a portion containing the top of
the pyramid and forming a recess in the base of the pyramid. The
recess in the base has a function of holding a specimen solution,
and the metal film 12 is formed on the recess of the base of the
dielectric block 11. Although not shown in FIG. 4, the polymer is
bound to the metal film 12, and the polyamino group and the
long-chain alkyl-based group are directly or indirectly bound to
the surface of the polymer.
[0089] In addition, the leakage-mode measurement system as
reported, for example, in Bunko Kenkyu (Journal of the
Spectroscopic Society of Japan, in Japanese), Vol. 47, No. 1, pp.
21-23 & 26-27, 1998 is another type of measurement system which
also utilizes the attenuated total reflection (ATR). Specifically,
the leakage-mode measurement system includes: a dielectric block
having a prismatic shape; a cladding layer formed on a face of the
dielectric block; an optical waveguide layer formed on the cladding
layer so that the optical waveguide layer can be in contact with a
specimen solution; a light source which generates a light beam; an
optical system which makes the light beam injected into the
dielectric block at various incident angles so that the light beam
is totally reflected at the boundary between the dielectric block
and the cladding layer; and a light detection unit which can detect
the state in which the attenuated total reflection occurs (i.e.,
the state in which the propagation mode is excited) by measuring
the intensity of the light beam totally reflected at the above
boundary. The biosensor chip according to the present invention can
also be used in such a leakage-mode measurement system.
[0090] Further, the biosensor chip according to the present
invention can also be used in a biosensor which has a waveguide
structure with a diffraction grating (and an additional layer in
some cases), and detects change in the refractive index by use of
the waveguide. The structures of the biosensor chips of this type
are disclosed in, for example, Japanese Examined Patent Publication
No. 6(1994)-027703, page 4, line 48 to page 14, line 15 and FIGS. 1
to 8 (and U.S. Pat. No. 5,071,248, columns 3 to 13 and FIGS. 1 to
8) and U.S. Pat. No. 6,829,073, column 6, line 31 to column 7, line
47 and FIGS. 9A and 9B. Furthermore, the biosensor chip according
to the present invention can be used in a biosensor having another
structure in which an array of grating-coupled waveguides are
incorporated within wells of a microplate as disclosed in Japanese
Unexamined Patent Publication No. 2007-501432 (corresponding to
U.S. Pat. No. 6,985,664). That is, in the case where the
grating-coupled waveguides are arrayed on the bottoms of the wells
of the microplate, the screening of a medical or chemical substance
can be performed with high throughput.
3. Evaluation of Examples
[0091] The present inventors have produced concrete examples of the
biosensor chip according to the present invention as indicated
below.
3.1 Concrete Example 1
[0092] The concrete example 1 of the biosensor chip according to
the present invention has been produced as follows.
[0093] A 1:1 mixture of a water solution containing 2.8 mM HODhbt
(3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine) and a water
solution containing 0.4M EDC (1-ethyl-3-(3-dimethylaminopropyl)
ethylcarbodiimide hydrochloride, available from Dojindo
Laboratories, Japan) is prepared, 100 ml of the mixture solution is
placed in contact with the surface of a Biacore sensor chip CM-5
(which is the research grade, and available from GE Healthcare
Bio-science KK, Japan), and a reaction with the mixture solution is
continued for ten minutes at room temperature. Then, the chip is
washed with water, and dried at room temperature in a vacuum drier
for ten minutes. Subsequently, 100 ml of
1,2-bis(2-aminoethoxy)ethane is placed in contact with the surface
of the chip, and a reaction with 1,2-bis(2-aminoethoxy)ethane is
continued for ten minutes at room temperature. Then, the chip is
washed with water. Thereafter, a solution of 1.0 g of stearic acid
(available from Signa-Aldrich Japan K.K.) and 0.1 g of Tween 20
(available from Tokyo Chemical Industries Co., Ltd.) in 1.0 ml of
DMF (dimethylformamide) is prepared, and 0.5 ml of a water solution
of 0.1M N-hydroxysuccinic acid imide (available from Dojindo
Laboratories, Japan) and 0.5 ml of a water solution of 0.4M EDC are
added to and mixed in 0.5 ml of the DMF solution of Tween 20 and
stearic acid and a reaction with the mixed solution is continued
for ten minutes at room temperature. Then, the chip is washed with
DMF, and thereafter with water. Thus, the concrete example 1 of the
biosensor chip according to the present invention has been
obtained.
3.2 Concrete Example 2
[0094] The concrete example 2 of the biosensor chip according to
the present invention has been produced in a manner similar to the
concrete example 1 except that oleic acid is used instead of
stearic acid.
3.3 Concrete Example 3
[0095] The concrete example 2 of the biosensor chip according to
the present invention has been produced as follows.
[0096] A 1:1 (v/v) mixture of a water solution containing 0.1 mM
NHS (N-hydroxysuccinamide) and a water solution containing 0.4M EDC
is prepared, and 100 ml of the mixture solution is placed in
contact with the surface of a Biacore sensor chip CM-5 (research
grade). Then, the chip is washed with water, and a reaction with a
solution of 0.1 mg of oleylamine in 1 ml of DMF is continued for
ten minutes at room temperature. Subsequently, the chip is washed
with water and dried. Thereafter, a 1:1 (v/v) mixture of a water
solution containing 2.8 mM HODhbt and a water solution containing
0.4M WSC (water-soluble carbodiimide) is prepared, 100 ml of the
mixture solution is placed in contact with the surface of the chip,
and a reaction with the mixture solution of HODhbt and WSC is
caused. Then, the chip is washed with water, and dried at room
temperature in a vacuum drier for ten minutes. Next, 100 ml of
1,2-bis(2-aminoethoxy)ethane is placed in contact with the surface
of the chip, and a reaction with 1,2-bis(2-aminoethoxy)ethane is
continued for ten minutes at room temperature. Then, the chip is
washed with water. Further, a solution of 0.1 mg of acetyl chloride
in 1.0 ml of DMF is prepared, and a reaction of with the DMF
solution of acetyl chloride is caused, and the chip is left unmoved
at room temperature for one hour. Then, the chip is washed with
DMF, and thereafter with water. Thus, the concrete example 3 of the
biosensor chip according to the present invention has been
obtained.
3.4 Concrete Example 4
[0097] The concrete example 4 of the biosensor chip according to
the present invention has been produced as follows.
[0098] A 1:1 mixture of a water solution containing 2.8 mM HODhbt
and a water solution containing 0.4M EDC is prepared, 100 ml of the
mixture solution is placed in contact with the surface of a Biacore
sensor chip CM-5 (research grade), and a reaction with the mixture
solution is continued for ten minutes at room temperature. Then,
the chip is washed with water, and dried at room temperature in a
vacuum drier for ten minutes. Subsequently, 100 ml of
1,2-bis(2-aminoethoxy)ethane is placed in contact with the surface
of the chip, and a reaction with 1,2-bis(2-aminoethoxy)ethane is
continued for ten minutes at room temperature. Then, the chip is
washed with water. Thereafter, a solution of 1.0 g of stearic acid
in 1.0 ml of DMF is prepared, and 0.5 ml of a water solution of
0.1M N-hydroxysuccinic acid imide and 0.5 ml of a water solution of
0.4M EDC are added to and mixed in the DMF solution of stearic
acid, and a reaction with the mixed solution is continued for ten
minutes at room temperature. Then, the chip is washed with DMF, and
thereafter with water. Further, a solution of 0.1 mg of acetyl
chloride in 1.0 ml of DMF is prepared, and a reaction of with the
DMF solution of acetyl chloride is caused, and the chip is left
unmoved at room temperature for one hour. Then, the chip is washed
with DMF, and thereafter with water. Thus, the concrete example 4
of the biosensor chip according to the present invention has been
obtained.
3.5 Measurement of Sensorgram
[0099] The present inventors have measured the absorbed amounts of
three types of liposomes (cationic, anionic, and nonionic
liposomes) by the biosensor chip as the concrete example 1 and the
Biacore sensor chip L1 (which is available from GE Healthcare
Bio-science KK, Japan, and used as a comparison example), where the
liposomes used in the measurement are ones of the liposomes
COATSOME which are available from NOF Corporation. In the Biacore
sensor chip L1, dextran arranged on a substrate is modified with a
long-chain alkane. (COATSOME is a registered trademark of NOF
Corporation.) In the measurement, each of the biosensor chip as the
concrete example 1 and the Biacore sensor chip L1 as the comparison
example is set on the surface plasmon resonance system Biacore 3000
(available from GE Healthcare Bio-science KK, Japan). FIGS. 5 and 6
show the sensorgrams which have been obtained by the above
measurement.
3.6 Measurement of Ability to Prevent Pollution
[0100] The present inventors have measured the ability to prevent
pollution of the biosensor chips as the concrete examples 1 to 4
and the Biacore sensor chip L1 as the comparison example on the
basis of absorption of methylene blue. In the measurement, each of
the biosensor chips as the concrete examples 1 to 4 and the Biacore
sensor chip L1 as the comparison example is set on the surface
plasmon resonance system Biacore 3000. The results of the
measurement, as well as the amounts of absorption of the cationic,
anionic, and nonionic liposomes, are indicated in Table 1, where
absorption of methylene blue from 0 to 2000 RU (resonance unit) is
indicated by a blank circle, and absorption of methylene blue
greater than 2000 RU is indicated by a cross.
TABLE-US-00001 TABLE 1 Ability to Absorbed Amount of Liposome
Protect Cationic Anionic Nonionic Pollution Concrete 8707 7636 7644
.largecircle. Example 1 Concrete 9358 8757 8530 .largecircle.
Example 2 Concrete 11688 9265 9852 .largecircle. Example 3 Concrete
8690 10067 10447 .largecircle. Example 4 Comparison 31860 2062 794
X Example
[0101] As understood from FIGS. 5 and 6 and Table 1, the amounts of
absorption of the anionic and nonionic liposomes by the sensor chip
as the comparison example are extremely small, and the amounts of
absorption greatly vary depending on the electric charge. On the
other hand, the biosensor chips as the concrete examples 1 to 4
absorb all of the cationic, anionic, and nonionic liposomes. That
is, the biosensor chip according to the present invention can
absorb liposomes regardlessly of the electric charge. Therefore,
the biosensor chip according to the present invention has great
versatility. In addition, as indicated in Table 1, the amount of
absorption of low-molecular-weight compounds by the biosensor chip
according to the present invention is small. That is, the biosensor
chip according to the present invention exhibits high ability to
protect pollution.
* * * * *